In utero electroporation of E-15 Swiss Webster mice (strain B6.129-Calb1tm1Mpin/J, The Jackson Laboratories, Bar Harbor, Maine) with plasmids expressing Fast-GCaMP6f variants and GCaMP6f under the CAG promoter yielded expression of the GECI in L2/3 pyramidal neurons. -thick cortical brain slices were prepared from P14-21 mice using ice-cold artificial CSF (aCSF) (126-mM NaCl, 3-mM KCl, 1-mM , 20-mM D-glucose, 25-mM , 2-mM and 1-mM ; saturated with 95% ). Slices were preincubated at 34°C for 1 h and then kept at room temperature. During recording and imaging, session slices were transferred to an immersion-type recording chamber perfused at 2 to with aCSF solution heated up to the physiological temperature () and saturated with 95% . Patch clamp recordings were obtained with borosilicate electrodes (6 to 9 MΩ) filled with a solution containing high potassium intercellular solution (133-mM methanesulfonic acid, 7.4-mM KCl, 0.3-mM , 3-mM , and 0.3-mM ; 290 mOsm; pH adjusted to 7.30 with KOH) using a shadowpatch technique. Electrophysiological signals were acquired with an Axopatch 200B amplifier and Clampex 8.0 software (Axon Instruments, Foster City, California). After whole-cell break-in, cells were held in current clamp mode (holding currents at , to ; series resistance 15 to 30 MΩ). Series resistance was monitored periodically and compensated by balancing the bridge. Spiking was induced through injection of current pulses at various amplitudes and durations and individual trials were separated by at least 10 s. L2/3 neurons were imaged using a custom-built two-photon laser scanning microscope using pulsed 830 nm (OGB-1) or 920 nm (GCaMP) excitation from a Ti:sapphire laser (Mira 900, Coherent). Excitation power was kept below 15 mW at the backplane of the objective (, NA 0.8 IR-Achroplan; Carl Zeiss, Thornwood, New York). Line scans (500 Hz) were made from dendrites at least 1 cell-diameter away from the soma. Data acquisition was controlled by ScanImage r3.6.1.